The first part of this activity will introduce the student to the use of mitochondrial DNA as a tool for familial identification. The fascinating story of the Romanov executions and subsequent late 1990's discovery of their gravesite is the basis of a self directed classroom activity. Students learn how mtDNA technology was used to rule out the possibility of Anna Anderson's claim of Romanov lineage. This unit also presents students with the opportunity to use Bioinformatics tools as a means to discover biological relationships. Students will use real Mitochondrial DNA data and Biology Workbench to compare DNA sequences. Through comparison of this data, students will be able to determine evolutionary relationships beteen pre-historic and modern human mitchondrial DNA.

BACKGROUND -- Humans actually carry a second set of genetic information in their mitochondria. All of your mitochondria come from your mother. Thus, your entire Mitochondrial DNA is an exact copy of your mother’s. Mitochondrial DNA is small and circular. Through analyzing a small region, approximately 444 base pairs, of Mitochondrial DNA we can determine evolutionary relationships. Since this portion of DNA does not code for any proteins it accumulates mutations at a faster rate then Nucleic DNA. Organisms with fewer mutations are more closely related then those with a greater number of mutations.

Go to the website http://www.dnai.org. Click on Recovering the Romanovs module, read the introduction, and click on The Romanov Family at the top of the page. You will be visiting each of the three sections of this application.Click on Start and proceed through each section using the forward arrow button at the bottom of the page. Be sure to visit each activity in the module for background information for the following activity.Activity

Through the use of a timeline, poster or written paper, demonstrate how scientists used mitochondrial DNA to disprove Anna Anderson’s claim to Romanov lineage.

ACTIVITIES IN SPECIAL SETTINGS- Computer Lab (Part II)

1) Go to the “Biology Workbench" - http://workbench.sdsc.edu (Follow the "Setup a free account" link to register as a user). 2) Obtain “Data Sheet” and “mtDNA” from “Uploaded Files” (Found at the very bottom of Inquiry page)3) After logging in, schroll down and click on “session tools”.4) Select “Start New Session” and then click “Run”.5) Give this session a title (Ex: Human mt DNA)6) Click “Start New Session”7) Click “Nucleic tools” tab from top of page8) From pull down menu select “Add New Nucleic Sequence” then click “Run”9) Under the sequence portion, cut and paste the given sequences (This is your "mtDNA" file that you already downloaded in #2)10) Click “Save” after pasting all the sequences11) On next page, be sure to check the Following boxes:a. YixiChina, EgyptianMummy, Otzi, LakeMungoManb. Select 1 of the 6 modern sequences.12) On scroll down portion, choose “CLUSTALW” then click “Run”13) Then click “Submit”.14) Answer questions #1&2!15) Click “Import Alignment”, then click “Run”.16) ***You are now in Alignment Tools!17) On scroll down, choose “Textshade”. Be sure to check box mlabeled "ClustalW - Nucleic" for this alignment. Then click “Run” and then “Submit” (Be patient this may take a few minutes)18) Answer Questions #3&4.19) On workbench page click “Return” button.20) On scroll down choose “DRAWGRAM” then click “Run” and then “Submit”.21) Draw Phenogram found on this page.22) Click “Return” button.23) From scroll down, choose “CLUSTALDIST” (Be sure “CLUSTALW” box is check on side).24) Click “Run” and then “Submit”25) Copy the “Clustal Distance Matrix” numbers down under the Phenogram that you drew. (This should go on chart found on your data sheet).a. Convert numbers to percentages (Hint: Multiply by 100).26) Answer questions #5-7.

Part IIn groups of 3 or 4 brainstorm other ways mitochondrial DNA might be used in society. Come up with at least two additional scenarios in which mtDNA might be used. Describe whose DNA would be needed in your scenario.

Part II

1.) What do the dashes mean on the “Sequence Alignment”? What does “No consensus” mean?

3.) Why do you think that the Egyptian has the most pink boxes in its sequence?

4.) What do you think caused the Egyptians to have more of these pink boxes?

5.) The “Clustal Distance Matrix” shows relationships between 2 organism’s mitochondrial DNA. The numbers themselves are percentage differences. What can you tell about 2 organisms who have a zero (0) or a very low percentage versus organisms with a high percentage?

6.) On your Phenogram, which organisms are the least similar? Most similar?

How could you use Biology Workbench to determine your ancestral lineage?

Credits & Acknowledgements

SEPCHE Institute for Mathematics and Science. Institute: Immaculata University. Course: Utilizing Bioinformatics to Integrate Technology into the Biology Classroom. Instructor: Sister Susan Cronin, IHM, Ph.D. The 2005 Summer Courses for Teachers is a SEPCHE Collaborative Project designed by the SEPCHE Committee of Chief Academic Officers and funded by a grant from the U.S. Department Housing and Urban Development.